Escherichia coli K1 is a leading cause of septicemia and meningitis in newborn infants. Frequent ineffectiveness of conventional antibiotic therapy indicates the need for better understanding of the genetic determinants underlying K1 colonization and invasion, which might suggest new modes of prophylaxis or treatment. Currently, little is known about how K1 colonizes and causes invasive disease, or about the specific mechanisms allowing transition from mucosal surface colonization to penetration of and growth in normally sterile host compartments. However, it is known that the closely related laboratory strain E. coli K-12 is unable to persist in either the intestinal tract or the bloodstream. Thus, information on the genetic determinants of colonization and invasion is contained in the differences between the closely related genomes of these two bacteria. In this project, a new technique of integrated structural and functional mapping of the genome (called "genetic clamping, "genomic silhouetting", or "genomic prospecting by genetic inference"), which exploits genetic map conservation between strains in order to detect chromosomal physical-distance discrepancies between them (Bloch, et al., J. Bacteriol., 1994; Rode, et al., Gene, 1995; Bloch, et al., Biochem. Biophys. Res. Com. 1996), is used to isolate K1-specific chromosomal segments containing K1 virulence genes (i.e., "pathogenicity islands"). First, new tools (vectors for introduction of minitransposons carrying ultrarare restriction sites) for accurate/general implementation of this method will be constructed (specific aim 1). Second, these tools will be used to construct a set of insertions circumscribing the chromosome of E. coli K-12 strain MG1655 (specific aim 2). Third, artificial macrorestriction fragments from double-insertion mutants of strain MG1655 and K1 strain RS218 (each pair of double mutants containing the identical pair of insertions) will be used to detect K1-specific (and K-12-specific) chromosomal segments (specific aim 3). Finally, the K1-specific segments will be characterized to determine their contributions to RS218 virulence (specific aim 4). This new integrated physical and genetic approach allows rapid and efficient isolation of pathogen-specific segments, which will ultimately be dissected to link in-vivo phenotypes to individual RS218 virulence loci. Thus, these studies are expected to lead to improved understanding and possibly more effective treatment of E. coli K1 newborn disease, and to new multilocus probes for effective-epidemiologic surveillance and prevention.